This invention relates to a method for inhibiting apoptosis in ischemic-reperfused myocardium. More specifically this invention relates to a method for using heparin or noncoagulant heparin in the prevention of apoptosis.
Cells die by one of either of two processes: necrosis or apoptosis. Whereas necrosis occurs through external injury producing cellular membrane destruction, swelling and lysis, apoptosis is endogenously mediated cellular suicide effected by activation of a series of aspartate-specific proteases called caspases and endonucleases, resulting in proteolytic destruction of cellular proteins and chromosomal elements. Apoptotic events include DNA fragmentation, chromatin condensation, membrane blebbing, cell shrinkage, and disassembly into membrane-enclosed vesicles (apoptotic bodies). In vivo, this process culminates with the engulfment of apoptotic bodies by other cells, preventing complications that would result from a release of intracellular contents. In myocardial infarction, both processes contribute to myocardial muscle injury and destruction. Overt necrosis predominates in the central zone of infarcted myocardium, and apoptosis occurs in the border zones of histologically infarcted myocardium. See, G. Olivetti, et al., xe2x80x9cAcute myocardial infarction in humans is associated with activation of programmed myocyte cell death in the surviving portion of the heart,xe2x80x9d J. Mol. Cell Cardiol, 28:2005-2016, 1994; and A. Saraste, et al., xe2x80x9cApoptosis in human myocardial infarction,xe2x80x9d Circulation, 95:320-323, 1997. Also, apoptosis occurs in hypoperfused hibernating myocardium. See, C. Chen, et al., xe2x80x9cMyocardial cell death and apoptosis in hibernating myocardium,xe2x80x9d J.A.C.C, 30:1407-1412, 1997. Apoptosis also contributes substantially to myocyte death in patients suffering heart failure from dilated cardiomyopathy. See, A. Haunstetter, et al., xe2x80x9cBasic mechanisms and implications for cardiovascular diseases,xe2x80x9d Circ. Res., 82:1111-1129, 1998.
Apoptosis is controlled at two distinct levels. First, cells have unique sensors, termed death receptors, on their membrane surface. Death receptors detect the presence of extracellular death signals and, in reponse, ignite the cell""s intrinsic apoptosis machinery. See, A. Ashkenazi, et al., xe2x80x9cDeath receptors: signaling and modulation,xe2x80x9d Science, 281:1305-1308, 1998. One of the more important receptors is the member of the tumor necrosis receptor family TNFR1 (also called p55). When tumor necrosis factor (TNF) attaches to TNFR1, the receptor trimerizes, and binds a series of other proteins: TRADD ((TNFR-associated death domain); TRAF-2 (TNFR-associated factor-2; RIP (receptor-interacting protein): and FADD (Fas-associated death domain). FADD couples the TNFR1-TRADD complex to activate caspase-8, thereby initiating activation of the entire cascade of other caspases that effect apoptosis. TNF plays an important role in ischemia-reperfusion injury and in the contractile depression of myocardium following ischemia and reperfusion during myocardial infarction. See, B. S. Cain, et al., xe2x80x9cTherapeutic strategies to reduce TNF-xcex1 mediated cardiac contractile depression following ischemia and reperfusion,xe2x80x9d J. Mol. Cell. Cardiol., 31:931-947. TNF plays an important role in hemorhagic shock. See, D. R. Meldrum, et al., xe2x80x9cHemorrhage activates myocardial NFxcexaB and increases TNF-xcex1 in the heart,xe2x80x9d J. Mol. Cell. Cardiol., 29:2849-2854, 1997. Apoptosis from TNF produced endogenously by overloaded myocardium also plays a significant role in mediating cardiac apoptosis leading to initiation and progression of congestive heart failure. See, for example, J. Narula, et al., Apoptosis in myocytes in end-stage heart failure,xe2x80x9d New England J. Med, 335:1182-1189, 1996; and T. Kubota, et al., et al., xe2x80x9cDilated cardiomyopathy in transgenic mice with cardiac-specific overexpression of tumor necrosis factor-xcex1,xe2x80x9d Circ. Res., 81:627-635, 1997.
At a second site, activation of caspases and subsequent apoptosis are initiated by events that disturb mitochondria. Either disruption of electron transport and aerobic oxidative phosphorylation or opening of pores in the outer mitochondrial membrane by pro-apoptotic cytoplasmic proteins of the BAX or BH3 families will allow leakage out of the mitochrondria of the respiratory chain component cytochrome c. Upon entering the cytoplasm, cytochrome c binds to a cytosolic protein called apoptotic protease activating factor-1 (Apaf-1). In the presence of ATP, the complex of cytochrome c and Apaf-1 activate procaspase 9, which initiates subsequent activation of the remainder of the caspase cascade and initiation of cellular apoptosis. See, D. R. Green, et. al., xe2x80x9cMitochrondria and apoptosis,xe2x80x9d Science, 81:1309-1312, 1998.
The death domain and mitochrondrial pathways of caspase and apoptosis activation are interrelated in that TNF can stimulate neutral membrane sphingomyelinase, resulting in production of ceramide, which disrupts mitochrondrial electron transport, also eventually effecting release into the cytoplasm of mitochondrial cytochrome c. Cytochrome c plays a prominent early role in the signal transduction of caspase activation and cardiomyocyte apoptosis induced by reactive oxygen species. See, R. von Harsdord, et al., xe2x80x9cSignaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis,xe2x80x9d Circ., 99:2934-2941, 1999. Production of reactive oxygen species is greatly enhanced as a consequence of ischemia-reperfusion of myocardium and oxidant stress produced during ischemia-reperfusion induces myocardial apoptosis. See, N. Maulik, et al., xe2x80x9cOxidative stress developed during the reperfusion of ischemic myocardium induces apoptosis,xe2x80x9d Free Rad. Biol. Med, 24:869-875, 1998. Thus, the activity of cytochrome c when it is transported to the cytoplasm appears to play an important and pivotal role in activating pro-apoptotic cascades, whether the initial induction of apoptosis is effected through membrane death receptor or mitochrondrial pathways.
In view of the foregoing it is readily apparent that there is a need for treatment of myocardial reperfusion injury that inhibits or prevents apoptosis.
It is therefore the general object of this invention to provides a method of inhibiting or preventing apoptosis in ischemic-reperfused myocardium using heparin or nonanticoagulant heparin.
The present invention provides a method for inhibiting apoptosis in ischemic-reperfused myocardium by administering to a mammal an effective amount of heparin to reduce myocardial cell death in myocardial infarction. It has been found that at doses greatly exceeding those needed for anticoagulation heparin substantially reduces reperfusion injury both in the isolated perfused heart and intact whole animal models of myocardial infarction. This protective effect is independent of heparin""s activity as an anticoagulant.
In accordance with another aspect of this invention, there is provided a method for inhibiting apoptosis in ischemic-reperfused myocardium by administering to a mammal an effective amount of nonanticoagulant heparin, such as O-desulfated heparin, to reduce or prevent myocardial cell death in myocardial infarction.
In yet another aspect of this invention it was found that heparin or nonanticoagulant heparin when conjugated to a lipophilic moiety such as a fatty acid or cholesterol by reaction across a carboxylic acid or free amine group can be used to enhance cellular uptake by cell types not normally concentrating heparin, such as neurons, thereby enhancing the anti-apoptotic effect of heparin or nonanticoagulant heparin. Furthermore, heparin or nonanticoagulant heparin, either alone or conjugated to a lipophilic group, can be used to block apoptosis in situations of acute trauma, such as generalized trauma, global ischemia-reperfusion injury occurring as a consequence of hemorrhagic shock, or spinal cord injury, thereby preventing cell death in organs such as the spinal cord.